Mutant proteinase with reduced self-cleavage activity and method of purification

ABSTRACT

The present invention provides a mutant 27 kDa NIa proteinase having reduced self-cleavage activity relative to the self-cleavage activity of its wild-type proteinase. The mutant has the same substrate cleavage activity as the wild-type proteinase but is more stable than the wild-type proteinase. The present invention also provides a method of obtaining large quantities of active 27 kDa NIa proteinase for use as a tool for purification of other proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/184,315, which isa continuation of U.S. Ser. No. 10/343,766, which is a 35 U.S.C. 371application of PCT/US01/18620 filed on Jun. 11, 2001, which claimsbenefit of U.S. Ser. No. 60/211,535 filed on Jun. 15, 2000. All of theseapplications are incorporated in their entirety by reference for allpurposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: YALE 040 03US Seq.txt,date recorded: May 24, 2012, file size 2 kilobytes).

FIELD OF THE INVENTION

The present invention is related to compositions comprising mutantproteinase having reduced self-cleavage activity relative to theself-cleavage activity of its wild-type proteinase. The mutantproteinase has the same substrate cleavage-activity as its wild-typeproteinase and it is more stable than its wild-type proteinase. Thepresent invention is also related to methods of obtaining largequantities of purified active proteins that form inclusion bodies incells.

BACKGROUND

Proteinases are present in both prokaryotic and eukaryotic systems andhave been shown to play an important role in the processing of largeprecursor polyproteins during viral replication. The reliance of viruseson proteolytic processing by virally encoded proteases has beensuggested to have several evolutionary advantages, such as a need forreduced genomic content and partial release from the constraints of themechanisms of transcriptional and translational regulation of the hostcell (Lawson et al., 1990). Viruses have evolved methods for regulatingthe proteolytic cascade that produces viral structural and replicationproteins to replace the host mechanisms. Lawson et al. (1991) reportthat in picornaviruses, the temporal and spatial distribution ofexpressed protease activity affects the appearance and location of thefinal proteolytic product.

Potyviruses are members of the picornaviral superfamily that infectplants. Like other members of the picornaviral family, potyviruses makeextensive use of proteinases during replication. An example of apotyvirus that possesses a genome encoding a single large polyproteinproteolytically processed by virally encoded protease is the tobaccoetch virus (TEV). TEV has a single-strand, plus-sense RNA genome ofabout 9,500 nucleotides. The RNA is organized as a single open readingframe and encodes a 346 kDa polyprotein (Allison et al., 1986). Thepolyprotein is co- and post-translationally processed by viral encodedproteinases. Two proteinases, P1 and helper component proteinase(HC-Pro), are responsible for their autocatalytic release from theamino-terminus of the polyprotein (Verchot et al. 1991; Carrington etal., 1989). The third proteinase, nuclear inclusion proteinase (Nia),mediates all other cleavage events.

Characterization of the 27 kDa NIa Proteinase of Potyvirus

The NIa proteinase is 49 kDa and is found as an aggregate with the 54kDa NIb polypeptide in nuclear inclusion bodies in infected plant cells(Carrington et al., 1988; Parks et al., 1995). The 49 kDa NIa proteinaseis a picornavirus 3 C-like proteinase that recognizes cleavage siteswithin the C-terminal two-thirds of the polyprotein. The proteolyticdomain of NIa lies within the C-terminal half of the protein and has amolecular weight of about 27 kDa, while the N-terminal region of NIacontains the Vpg (viral protein, genome-linked) activity and has amolecular weight of about 21 kDa.

Structurally, the 27 kDa NIa proteinase has been reported to be similarto the trypsin-like family of cellular serine proteinases, such aschymotrypsin or trypsin, with the substitution of Cys for serine as theactive site nucleophile (Blazan at al., 1990; Dougherty at al., 1989).Dougherty et al. (1989) disclose that the catalytic triad of 27 kDaproteinase is composed of His, Asp, and Cys, being similar to thecatalytic triad found in other viral proteinases (Dougherty et al.,1989). However, unlike the other proteinases, the 27 kDa proteinaserecognizes an extended heptapeptide sequence, E-X-X-Y-X-Q↓S/G (positionsP6-P1↓P′1; X is any amino acid) (SEQ ID NO: 1), and cleaves within theheptapeptide sequence (Dougherty at al., 1989a; Dougherty et al., 1988;Dougherty et al., 1989b). Residues at positions P6, P3, P1, and P′1 areconserved and essential for optimal cleavage. Amino acids at the otherpositions appear to modulate the rate at which cleavage occurs(Dougherty et al., 1989; Dougherty and Parks, 1989).

Moreover, the 27 kDa NIa proteinase appears to be structurally andfunctionally similar to other plus-stranded RNA viral-encodedproteinases (Krausslich and Wimmer, 1998). First of all, it cleaves thepolyprotein between particular Gln-Gly or Glyn-Ser dipeptides. Secondly,proteolytic activity is enhanced by dithiothreitol. Thirdly, the geneencoding this proteinase is adjacent to the putative RNA-dependent,RNA-polymerase gene. Lastly, the proteinase contains a conservedC-terminal amino acid motif (Cys-˜15 amino acids—His) (Argos et al.,1984). This last characteristic is shared by proteinases encoded by manyRNA viruses that translationally express their genetic information as asingle polyprotein from genome length RNA (Dougherty et al., 1989).

Additionally, Parks at al. (1995) report that the 27 kDa NIa proteinasecontains an internal self-cleavage site positioned at 24 amino acidsfrom the carboxyl terminus of the proteinase and that the active 27 kDaproteinase converts to a lower molecular weight form with time. The 27kDa NIa proteinase lacking the C-terminal 24 amino acids exhibitslimited activity. The truncated proteinase is about one-twentieth asefficient in proteolysis of a test peptide substrate as the full lengthform, and Parks at al. (1995) indicate that the 27 kDa NIa proteinaseappears to lose its activity with time.

Further, Polayes et al. (1994) disclose that the 27 kDa NIa proteinaseis a highly specific protease that is active under a broad temperaturerange and on a variety of substrates. Polayes et al. report rapidcleavage at 30° C. and 37° C., about 80% cleavage at both 21° C. and 16°C. in one hour, and 50% cleavage at 4° C. Accordingly, Polayes at al.recommend the use of this proteinase as a tool for removing affinitytags from fusion proteins.

Use of 27 kDa a Proteinase Cleavage System for Purification of Proteins

Parks et al. (1994) disclose an improved method for the production,cleavage, and purification of fusion proteins and peptides using the 27kDa NIa proteinase. The method comprises producing a fusion proteincomprising the protein of interest, a carrier peptide (such as anaffinity carrier) and a 27 kDa NIa proteinase cleavage site insertedbetween the two, purifying the fusion protein, and incubating the fusionprotein with the 27 kDa NIa proteinase to remove the carrier peptidefrom the protein of interest.

Johnston et al., U.S. Pat. No. 5,532,142, disclose a similar method ofisolation and purification of recombinant proteins using the 27 kDa NIaproteinase. Like the purification method of Parks et al. (1994), themethod of Johnston et al. involves producing large quantities of thefusion protein containing a desired protein fused to the 27 kDa NIaproteinase cleavage site which is the carrier peptide, purifying thefusion protein, and incubating the purified fusion protein with the 27kDa NIa proteinase to remove the carrier peptide from the desiredprotein.

Unlike other proteinases, the 27 kDa proteinase exhibits highspecificity, insensitivity to many proteinase inhibitors used in proteinpurification, and efficient cleavage under a broad range of temperatures(Polayes et al., 1994). Moreover, the protein of interest is easilyseparated from the carrier peptide and the 27 kDa proteinase. For thesereasons, there is an on-going interest in obtaining large quantities ofthe active protein for use as a tool in protein purification.

Purification of Proteins Including the 27 kDa NIa Proteinase fromInclusion Bodies

Purification of Proteins that Form Inclusion Bodies

The development of recombinant DNA technology has enabled the cloningand expression of proteins in bacteria, yeast and mammalian cells andhas made it possible to produce therapeutics and industrially importantproteins at economically feasible levels. However, the expression ofhigh levels of recombinant proteins in Escherichia coli often results inthe formation of inactive, denatured protein that accumulates inintracellular aggregates known as insoluble inclusion bodies (Krueger etal., “Inclusion bodies from proteins produced at high levels inEscherichia coli,” in Protein Folding, L. M. Gierasch and P. King (Eds),Am. Ass. Adv. Sci., 136-142 (1990); Marston, Biochem. J. 240:1-12(1986); Mitraki et al., Bio/Technol. 7: 800-807 (1989); Schein,Bio/Technol. 7:1141-1147 (1989); Taylor et al., Bio/Technol. 4: 553-557(1986)). Inclusion bodies are dense aggregates, which are 2-3 m indiameter and largely composed of recombinant protein, that can beseparated from soluble bacterial proteins by low-speed centrifugationafter cell lysis (Schoner et al., Biotechnology 3:151-154 (1985)).

The recovery of recombinantly expressed protein in the form of inclusionbodies has presented a number of problems. First, although the inclusionbodies contain a large percentage of the recombinantly produced protein,additional contaminating proteins must be removed in order to isolatethe protein of interest. Second, the proteins localized in inclusionbodies are in a form that is not biologically active, presumably due toincorrect folding.

Several methods have been developed to obtain active proteins frominclusion bodies. These strategies include the separation andpurification of inclusion bodies from other cellular components,solubilization and reduction of the insoluble material, purification ofsolubilized proteins and ultimately renaturation of the proteins andgeneration of native disulfide bonds. The art teaches thatconcentrations of 6 M or greater of chaotropic agents, such as guanidinehydrochloride, guanidine isothiocyanate or urea—are necessary forsolubilization of the insoluble recombinant polypeptides from theinclusion bodies. See, for example, Vandenbroeck et al, Eur. J. Biochem.215:481-486 (1993); Meagher et al., Biotech. Bioeng. 43:969-977 (1994);Yang et al., U.S. Pat. No. 4,705,848, issued Nov. 10, 1987; Weir et al.,Biochem. J. 245:85-91 (1987); and Fischer, Biotech. Adv. 12:89-101(1994). However, the use of high concentration of chaotropic agents,such as guanidine hydrochloride, to solubilize proteins denatures theproteins.

U.S. Pat. No. 5,912,327 discloses the use of low concentrations ofguanidine salts, about 0.7 to about 3.5 M, to solubilize biologicallyactive (i.e., correctly folded) proteins and extract this population ofthe protein from a heterogenous protein mixture localized in inclusionbodies. The method described in the patent comprises releasing theinclusion bodies containing the target protein from the cells by lysis,optionally washing the cells to remove cellular components, extractingwith solutions containing low concentrations of guanidine salts,refolding target proteins which have been solubilized using guanidinesalts by rapid dilution of guanidine salt extracts and optionallyemploying agents which facilitate target protein refolding. The proteincan then be recovered and purified by methods well known to the skilledartisan. However, this method is labor intensive.

Tissue plasminogen activator (tPA or TPA) is one example of apharmaceutically important drug produced by recombinant methods.Unfortunately the current methods for producing tPA from bacterial cellculture are both costly and laborious. The production of tPA inheterologous host organisms relies on the production of inactive tPAintracellularly in inclusion bodies, and the subsequent isolation andpurification of such inclusion bodies, followed by activation of the tPAonce freed from the inclusion bodies. U.S. Pat. No. 5,077,392 disclosesa renaturation method for refolding denatured proteins obtained afterexpression in inclusion bodies. tPA is isolated as a denatured reducedprotein and on subsequent oxidation refolded under oxidizing conditionsto obtain what was reported as up to a 26% yield of “reactivated”protein. While the method appeared to improve polypeptide yield, theprocess involves multiple, time-consuming steps, due to the initialrecovery of the insoluble, inactive protein.

Purification of 27 kDa NIa Proteinase.

The 27 kDa NIA proteinase has been especially difficult to isolate andpurify in large quantities and in active form because of its proclivityto form inclusion bodies in nature. Previously published purificationprotocols of TEV nuclear inclusion bodies from infected plant tissuehave demonstrated considerable proteolytic activity (Dougherty et al.,1980). However, attempts to separate the 49 kDa NIa proteinase from theNib protein and other components and to purify the NIa proteinase haveresulted in loss of protein activity (Parks et al., 1995).

Parks et al. (1995) describe purification of the soluble fraction ofrecombinantly produced 27 kDa NIa proteinase. The purification method ofParks et al. involves overexpressing the recombinant form of theproteinase as a fusion protein comprising a seven-His tag at theN-terminus and purifying the fusion protein using two separate columns,a nickel-nitrilotriacetic acid-agarose (Ni-agarose) column and acation-exchange column. This method is labor-intensive and producesinsufficient quantities of proteinase for use as a general tool inprotein purification.

Johnston et al., U.S. Pat. No. 5,532,142, discloses recombinant vectorsfor overproducing plant virus proteinases in suitable hosts. Johnston etal. use the same purification protocol as that of Parks et al. (1995) topurify the 27 kDa NIa proteinase. The yield of purified proteinase istypically in the range of 5 mg/liter of cell culture and not all of itis active. Thus, the yield of active protein is very low.

As discussed above, the 27 kDa NIA proteinase contains an internalself-cleavage site that when cleaved, produces a proteinase with reducedsubstrate cleavage activity. At the present, there is no known method ofstabilizing the proteinase, and there is no known method of obtaininglarge quantities of purified active 27 kDa NIa proteinase in largequantities. Accordingly, there is a need to develop a method ofobtaining large quantities of purified active 27 kDa proteinase thatwill not cleave itself.

SUMMARY OF THE INVENTION

The present invention provides mutant proteinases having a molecularweight of about 27 kDa and reduced self-cleavage activity relative tothe self-cleavage activity of its wild-type proteinase. In oneembodiment, the mutant proteinases of the present invention comprise anamino acid sequence in which the residue corresponding to Ser 219 of thewild-type 27 kDa NIa proteinase is replaced with another residue. Inanother embodiment, the mutant proteinases of the present inventioncomprises an amino acid sequence in which the residue corresponding toSer 219 is replaced with Asn.

The present invention also provides composition comprising the mutantproteinase. Preferably, the composition comprises a carrier in additionto the mutant.

Moreover, the present invention provides fusion proteins comprisingmutant proteinase having a molecular weight of about 27 kDa and reducedself-cleavage activity, fused to a heterologous polypeptide, fusionpartner, or carrier protein. In one embodiment, the heterologouspolypeptide, fusion partner, or carrier protein comprises a protein thatfacilitates its isolation. In a preferred embodiment, the heterologouspolypeptide consists of six histidines.

The present invention includes nucleic acid molecules comprising asequence encoding a mutant proteinase having a molecular weight of about27 kDa and reduced self-cleavage activity relative to the self cleavageactivity of to its wild-type proteinase. In one embodiment, the nucleicacid molecules encode mutant proteinases comprising an amino acidsequence in which the residue corresponding to Ser 219 of the 27 kDa NIaproteinase is replaced with another amino acid. In a preferredembodiment, the nucleic acid molecules encodes mutant proteinasescomprising an amino acid sequence in which the residue corresponding toSer 219 is replaced with Asn.

The present invention also includes vectors, expression vectors, andhost cells comprising a nucleic acid molecule encoding a mutantproteinase having a molecular weight of about 27 kDa and reducedself-cleavage activity relative to the self-cleavage activity of itswild-type proteinase.

Further, the present invention includes nucleic acid molecules encodingfusion proteins comprising a proteinase, having a molecular weight ofabout 27 kDA and reduced self-cleavage activity relative to the selfcleavage activity of its wild-type proteinase, fused to a heterologousprotein.

The present invention provides methods of producing a proteinase havinga molecular weight of about 27 kDa and reduced self-cleavage relative tothe self-cleavage activity of its wild-type proteinase comprisingcultivating a host cell comprising a nucleic acid encoding theproteinase under conditions that allow expression of the proteinase.

The present invention also provides a method of purifying a polypeptidethat forms inclusion bodies in a cell comprising:

-   -   a) obtaining cells expressing the polypeptide;    -   b) lysing the cells;    -   c) pelleting inclusion bodies by centrifugation of the lysed        cells;    -   d) centrifuging the inclusion bodies;    -   e) solubilizing the inclusion bodies in buffer containing        solubilization agent;    -   f) centrifuging the solubilized inclusion bodies to obtain a        supernatant containing the polypeptide;    -   g) loading the supernatant over a single denaturing column;    -   h) collecting the proteinase; and    -   i) renaturing the collected polypeptide.

In one embodiment, the polypeptide to be purified is a proteinase havinga molecular weight of about 27 kDa and reduced self-cleavage activityrelative to the self-cleavage activity of its wild-type proteinase. Inanother embodiment, the polypeptide is selected from the groupconsisting of tobacco etch virus (TEV) 27 kDa NIa proteinase and mutant27 kDa NIa proteinase. In a preferred embodiment, the polypeptide is themutant TPSN 27 kDa NIa proteinase, wherein the residue corresponding toSer 219 of the wild-type 27 kDa NIa proteinase is replaced with Asn.

Contemplated denaturing column of step (g) includes column containingNi-NTA resin. Contemplated methods of lysing the cells includefreeze-thaw cycles, sonication, and other enzymatic and mechanicalmeans. The present method also contemplates the addition of proteinaseinhibitors, preferably but not limited to PMSF, leupeptin, and pepstatinA, to the cells before lysis. The present invention includes the use ofbuffers, solubilizing agent, renaturing agents well-known to the skilledartisan for purifying proteins.

Additionally, contemplated methods of using the proteinase having amolecular weight of 27 kDa and reduced self-cleavage activity, includecleavage of substrate. In one embodiment of using the proteinase, theproteinase is incubated with a protein for a sufficient amount of timeto allow cleavage of the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows 27 kDa NIa proteinase purification—soluble vs insolublepreps. Samples from the soluble and insoluble purifications of TPWT(wild-type 27 kDa NIa proteinase) and TPSN (mutant form with Ser→Asnmutation) show that most of the 27 kDa NIa proteinase was expressed inthe insoluble fraction. Comparison of the final products from thedifferent purifications shows that insoluble preparations producegreater yields with higher purity than soluble preparations. Overloaded,renatured TPWT and TPSN samples demonstrate sample purity as compared toa commercially available 27 kDa NIa proteinase.

FIG. 2 shows specific activity for substrate cleavage by 27 kDa NIaproteinase. Specific activity is defined as picomoles substrate cleavedper picomole 27 kDa NIa proteinase in 1 hour at 30° C. TPWT and TPSNwere compared to a commercially available 27 kDa NIa proteinase(GibcoBRL) at several substrate concentrations. The specific activityappears to be comparable for all samples at the substrate concentrationstested.

FIG. 3 shows self-cleavage of 27 kDa NIa proteinase after a 5.5 weekincubation. TPWT, TPSN, and a commercially available 27 kDa NIaproteinase (GibcoBRL) were incubated at various temperatures for 5.5weeks at a concentration of approximately 187.5 g proteinase/ml(micrograms proteinase/ml) reaction. Comparison of the 4° C. and −20° C.truncated 27 kDa NIa proteinase bands to those in the −80° C. samplesindicates that self-cleavage activity is present in all samples at 4°C., but is absent from the TPSN sample at −20° C.

FIGS. 4A and 4B show self-cleavage of 27 kDa NIa proteinase at 4° C. and−20° C.

TPWT, TPSN, and a commercially available 27 kDa NIa proteinase(GibcoBRL)were incubated at various temperatures for 5.5 weeks. Aliquots removedat the timepoints shown were run on SDS-PAGE gels and quantitated usinga gel documentation system. Data were reported as the percent intensityof the truncated 27 kDa NIa proteinase band compared to the totalintensity of both the full-length and truncated bands. The graphs showthat TPWT has comparable self-cleavage activity to the commerciallyavailable 27 kDa NIa proteinase (assumed to have a wild typeself-cleavage site) at both 4° C. and −20° C., while TPSN has reducedself-cleavage activity at 4° C. and no self-cleavage activity at −20° C.

DETAILED DESCRIPTION OF TI-IE INVENTION

A. General Description

The present invention is based on the unexpected discovery that a singleamino acid change in the internal self-cleavage site will inhibitself-cleavage activity of the 27 kDa NIa proteinase. Mutating Ser atposition 219 of the wild-type 27 kDa NIa proteinase to Asn prevents theproteinase from cleaving itself.

The present invention relates to expression, overproduction, andpurification of virus proteinase. Sources of virus proteinase includeflavi-, picorna- and potyviruses. The present method is consideredparticularly suited for overproducing potyvirus proteinases in E. colior yeast cells.

The present invention is based on the development of a single-columnpurification method for isolation of milligram quantities of >95% pure,active 27 kDa NIa proteinase from inclusion body preparation. The newlydeveloped purification method yields about 32 mg of total pure proteinfrom the inclusion body preparation and greater than about 12 mg isactive.

Accordingly, the present invention provides a method of obtaining largequantities of active 27 kDa NIa proteinase from inclusion bodypreparation. The present invention also provides a mutant 27 kDa NIaproteinase having the same substrate cleavage as the wild-type 27 kDaNIa proteinase and a reduced self-cleavage activity. Thus, the presentinvention provides a 27 kDa NIa proteinase with increased shelf-life.

B. Definitions

Unless defined otherwise, all technical and scientific terms used inthis specification shall have the same meaning as commonly understood bypersons of ordinary skill in the art to which the present inventionpertains.

As used herein, “carrier” in a composition refers to a diluent,adjuvant, excipient, or vehicle with which the product is mixed.

As used herein, “carrier peptide or protein”, “fusion partner”, or“heterologous protein or polypeptide” of a fusion protein refers to theportion of the fusion protein that is added to the protein of interestfor the purpose of purification, for stability in production, or forother reasons.

As used herein, “control sequence or element” or “regulatory sequence orelement” refers to those non-translated regions of the vector, such asenhancers, promoters, 5′ and 3′ untranslated region, which interact withhost cellular proteins to carry out transcription and translation.

As used herein, “homologs” refers to proteins having the same or similarfunctions, especially proteins from different species having the same orsimilar functions.

As used herein, “inclusion body” refers to distinctive structuresfrequently formed in the nucleus or cytoplasm in cells infected withcertain filtrable viruses. They may be demonstrated by means of variousstains.

As used herein, “internal self-cleavage activity” or “internalautocatalytic activity” of a protein refers to the activity of cleavingat some site within the protein itself.

As used herein, “internal self-cleavage site” or “internal autocatalyticsite” is the site where the protein cleaves itself.

As used herein, “isolated nucleic acid” refers to a nucleic acid thathas been separated from its naturally occurring environment.

As used herein, “isolated polypeptide or protein” refers to apolypeptide or protein that has been separated from its naturallyoccurring environment.

As used herein, “proteinase” and “protease” are interchangeable termsand refer to enzymes that hydrolyze (break) polypeptide chains.

As used herein, “mutant proteinase” is a proteinase obtained by alteringthe nucleic acid encoding the wild-type proteinase and expressing thealtered nucleic acid. A “mutant” is a phenotype in which a mutation ismanifested. A “mutation” is a change in the chemistry of a nucleic acidthat is perpetuated in subsequent divisions of the cell in which itoccurs.

As used herein, “specific activity” is defined as picomoles of substratecleaved per picomole of proteinase in one hour at 30° C.

As used herein, “substrate cleavage activity” refers to the activity ofa proteinase in cleaving a specific amino acid sequence with a specificamount of activity.

As used herein, “wild-type proteinase” is a naturally occurringproteinase.

As used herein, “27 kDa NIa proteinase or protease” refers to aproteinase having a molecular weight of about 27 kDa and having the samesubstrate cleavage activity as the wild-type 27 kDa NIa proteinaseobtained from tobacco etch virus.

C. Specific Embodiments

1. Nucleic Acids Encoding Proteinases

The present invention provides nucleic acid molecules encodingproteinases having the same substrate cleavage activity as the wild-type27 kDa NIa proteinase, and preferably the nucleic acid molecules are inisolated form. In one embodiment, nucleic acid molecules provided by thepresent invention encode a proteinase having 27 kDa and reducedself-cleavage activity as compared to the self-cleavage activity of itswild-type proteinase. In another embodiment, nucleic acid molecules ofthe present invention encode a mutant form of the 27 kDa NIa proteinasecomprising an amino acid substitution corresponding to position 219 ofits wild-type proteinase, preferably Ser substituted with Asn, havingthe same substrate cleavage activity as the wild-type proteinase andhaving reduced self-cleavage activity relative to its wild-typeproteinase.

The nucleic acid molecules of the invention include deoxyribonucleicacids (DNAs), both single- and double-stranded deoxyribonucleic acids.However, they can also be ribonucleic acids (RNAs), as well as hybridRNA:DNA double-stranded molecules. Contemplated nucleic acid moleculesalso include genomic DNA, cDNA, mRNA, and antisense molecules. Thenucleic acids molecules of the present invention also include native orsynthetic, RNA, DNA, or cDNA, that encode a proteinase protein, or thecomplementary strand thereof, including but not limited to nucleic acidfound in a proteinase expressing organism, such as the tobacco etchvirus.

The nucleic acid sequence encoding the proteinase can be, for instance,substantially or fully synthetic. See, for example, Goeddel et al.,Proc. Natl. Acad. Sci. USA, 76, 106-110, 1979. For recombinantexpression purposes, codon usage preferences for the organism in whichsuch a nucleic acid is to be expressed are advantageously considered indesigning a synthetic proteinase-encoding nucleic acid. Codon usagepreferences for different organisms are well known to the skilledartisan. Since the nucleic acid code is degenerate, numerous nucleicacid sequences can be used to create the same amino acid sequence.

The nucleic acid molecules of the present invention can encodeproteinases having the same substrate cleavage activity as a wild-type27 kDa NIa proteinase. For example, the nucleic acid molecules canencode a proteinase having 27 kDa and reduced self-cleavage activity ora mutant form of the 27 kDa N7a proteinase having the same substratecleavage activity and having reduced self-cleavage activity as comparedwith its wild-type proteinase, piconaviral 3C proteinases, cellularserine proteinases such as chymotrypsin trypsin proteinases, proteinasesthat have internal self-cleavage sites similar to the internalself-cleavage site of the 27 kDa NIa proteinase, mutant forms of suchproteinases with reduced self-cleavage activity as compared to itswild-type proteinase, and proteinases that recognize the same extendedseven amino acid sequence (E-X-X-Y-X-Q↓S/G) (SEQ ID NO: 1) substratecleavage site as the 27 kDa NIa proteinase.

In one embodiment, the nucleic acids of the present invention encode aproteinase having 27 kDa and reduced self-cleavage activity as comparedto its wild-type proteinase. In another embodiment, the nucleic acids ofthe present invention encode a mutant form of the 27 kDa NIa proteinasecomprising an amino acid sequence in which the residue corresponding to219 of the 27 kDa NIa proteinase is replaced with another residue,preferably Asn.

Nucleic Acid Molecules Encoding Mutant Forms and Allelic Forms ofProteinases.

To construct mutant forms of proteinases having the same substratecleavage activity as the wild-type 27 kDa NIa proteinase and havingreduced self-cleavage activity, the nucleic acid encoding the wild-typeproteinase can be used as a starting point and modified to form thedesired mutants. For example, in the preferred embodiment, the nucleicacid sequence encoding the wild-type 27 kDa NIa proteinase is mutatedsuch that the Ser corresponding to position 219 in the encoded aminoacid sequence is replaced with another amino acid, preferably Asn.

Further, with an altered amino acid sequence, numerous methods are knownto delete sequence from or mutate nucleic acid sequences that encode apolypeptide and to confirm the function of the polypeptides encoded bythese deleted or mutated sequences. Accordingly, the invention alsorelates to a mutated or deleted version of a proteinase nucleic acidthat encodes a proteinase that has the same substrate cleavage activityas the wild-type 27 kDa NIa proteinase.

Conservative variants of the wild-type 27 kDa NIa proteinases or itsnaturally occurring isoforms and homologs are encompassed by the presentinvention. Such conservative mutations include mutations that switch oneamino acid for another within one of the following groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Gluand Gln;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

The types of substitutions selected may be based on the analysis of thefrequencies of amino acid substitutions between homologous proteins ofdifferent species developed by Schulz et al., Principles of ProteinStructure, Springer-Verlag, 1978, pp. 14-16, on the analysis ofstructure-forming potentials developed by Chou and Fasman, Biochemistry13, 211, 1974 or other such methods reviewed by Schulz et al.,Principles in Protein Structure, Springer-Verlag, 1978, pp. 108-130, andon the analysis of hydrophobicity patterns in proteins developed by Kyteand Doolittle, S. Mol. Biol. 157: 105-132, 1982.

The present invention also contemplates nucleic acids encoding naturallyoccurring allelic variants of the proteinases described above. In apreferred embodiment, allelic variants even though possessing a slightlydifferent amino acid sequence than the naturally occurring wild-type 27kDa proteinase will have the requisite ability to recognize and cleavethe heptapeptide sequence E-X-X-Y-X-Q1S/G (SEQ ID NO: 1). The presentinvention also contemplates conservative variants that do not affect theability of the proteinase to recognize and cleave the heptapeptidesequence E-X-X-Y-X-Q↓S/G (SEQ ID NO: 1). The present invention includes27 kDa NIa proteinase with altered overall charge, structure,hydrophobic/hydrophilic properties by amino acid substitutions,insertions, or deletions but still possess the ability to recognize andcleave the heptapeptide.

Preferably, the nucleic acids will encode proteinases having the samesubstrate cleavage activity as the wild-type 27 kDa NIa proteinase andhaving at least about 70% sequence identity, more preferably, at leastabout 80% sequence identity, even more preferably, at least about 85%sequence identity, yet more preferably at least about 90% sequenceidentity, and most preferably at least about 95% sequence identity to awild-type 27 kDa NIa proteinase or other naturally occurring isoformshaving the same substrate cleavage activity.

Numerous methods for determining percent homology are known in the art.One preferred method is to use version 6.0 of the GAP computer programfor making sequence comparisons. The program is available from theUniversity of Wisconsin Genetics Computer Group and utilizes thealignment method of Needleman and Wunsch, J. Mol. Biol. 48, 443, 1970,as revised by Smith and Waterman Adv. Appl. Math. 2, 482, 1981. Numerousmethods for determining percent identity are also known in the art, anda preferred method is to use the PASTA computer program, which is alsoavailable from the University of Wisconsin Genetics Computer Group.

Additionally, the invention includes substantially pure nucleic acidsthat hybridize under stringent conditions to a nucleic acid encoding aproteinase having the same substrate cleavage activity as the wild-type27 kDa NIa proteinase. Stringent hybridization conditions are conditionsin which hybridization to a labeled known nucleic acid sequence yields aclear and detectable sequence. Stringent conditions are those that (1)employ low ionic strength and high temperature for washing, for example,with 0.015 M NaCl, 0.0015 M sodium titrate, 0.1% SDS at 50° C.; (2)employ during hybridization a denaturing agent such as formamide, forexample, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1%Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; and (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., followed with washes at 42° C. in 0.2×SSC and 0.1% SDS. Askilled artisan can readily determine and vary the stringency conditionsappropriately to obtain a clear and detectable hybridization signal.

Nucleic Acid Molecules Encoding Fusion Proteins

The nucleic acid molecules of the present invention also encode fusionproteins comprising a proteinase such as those described in the previousparagraphs fused to a heterologous protein or polypeptide. In oneembodiment, the fusion proteins of the present invention comprise theproteinase having 27 kDa and reduced self-cleavage activity as comparedto its wild-type proteinase fused to a heterologous protein. In anotherembodiment, the fusion protein comprises the mutant 27 kDa NIaproteinase comprising an amino acid sequence in which the residuecorresponding to Ser 219 of the 27 kDa NIa proteinase is replaced withanother residue, preferably Asn, fused to a heterologous protein.

Nucleic acids encoding various heterologous proteins may be fused to thenucleic acid encoding the proteinase. For example, nucleic acid encodingaffinity tags such as the His tags, antibodies, or carrier peptides orproteins having specific binding properties such as Staphylococcusaureus protein A and the carbohydrate recognition domain (CRD) ofgalactose-specific rat hepatic lectin (Taylor and Drickamer, 1991), maybe fused to the proteinase for purification of the proteinase byaffinity chromatography. Nucleic acid encoding substrate cleavage sitesof thrombin or factor X may be fused to the nucleic acid encoding theproteinase for ease of removal of carrier peptide. Additionally, fusionproteins tend to be more soluble than a single protein, contributing tohigher yields and simpler purification. The fusion partner for theproteinase may be selected on the basis of transport characteristics toassure that the fusion protein is secreted into either the periplasmicspace or the growth medium. Moreover, the fusion partner may also beselected for increasing the stability of the proteinase in the preferredexpression system for obtaining large quantities of the protein. Forexample, fusion proteins are usually more stable in bacteria than thenative eukaryotic proteinase.

Nucleic Acid Encoding Proteins or Polypeptides Expressed in InclusionBodies.

Nucleic acids encoding proteins, polypeptides, or fusion proteins orpolypeptides that form inclusion bodies in cells are also encompassed bythe present application. Such nucleic acids can be expressed in hostcells to produce large quantities of the proteins or polypeptides ininclusion bodies, as discussed below. The proteins or polypeptides arethen purified by the purification method provided by the presentinvention, also discussed below.

2. Polypeptides or Proteins

Polypeptides of the invention include all proteinases having the samesubstrate cleavage activity as the wild-type 27 kDa NIa proteinase, andpreferably in isolated or purified form. The present invention alsoincludes these proteinases in native or synthetic form, including butnot limited to polypeptides purified from a proteinase-expressingorganism. In one embodiment, the polypeptides of the present inventioncomprise a proteinase having 27 kDa and reduced self-cleavage activityas compared to its wild-type proteinase. In another embodiment, thepolypeptides of the present invention comprise a mutant form of thewild-type 27 kDa NIa proteinase having the same substrate cleavageactivity as the wild-type proteinase, but with reduced self-cleavageactivity as compared to its wild-type proteinase, and having amino acidcorresponding to position 219 of the wild-type NIa proteinasesubstituted with another amino acid, preferably Asn.

Structurally, the 27 kDa NIa proteinase has been reported to be similarto the trypsin-like family of cellular serine proteinases, such aschymotrypsin or trypsin, with the substitution of Cys for serine as theactive site nucleophile (Blazan et al., 1990; Dougherty et al., 1989).Dougherty et al. (1989) discloses the catalytic triad of 27 kDa NIaproteinase to be composed of His, Asp, and Cys which is similar to thecatalytic triad found in other viral proteinases (Dougherty et al.,1989). However, unlike the other proteinases, the 27 kDa proteinaserecognizes an extended heptapeptide sequence, E-X-X-Y-X-Q↓S/G (positionsP6-P1↓P′1; X is any amino acid) (SEQ ID NO: 1), and cleaves within theheptapeptide sequence (Dougherty et al., 1989a; Dougherty et al., 1988;Dougherty et al., 1989b).

Proteinases having the same substrate cleavage activity as a wild-type27 kDa NIa proteinase include but are not limited to polypeptidescomprising the wild-type 27 kDa NIa proteinase or a mutant form thereofhaving the same substrate cleavage activity and having reducedself-cleavage activity as compared with its wild-type proteinase,piconaviral 3C proteinases, cellular serine proteinases such aschymotrypsin trypsin proteinases, proteinases that have internalself-cleavage sites similar to the internal self-cleavage site of the 27kDa NIa proteinase, mutant forms of such proteinases with reducedself-cleavage activity as compared to its wild-type proteinase, andproteinases that recognize the same extended seven amino acid sequence(E-X-X-Y-X-Q↓S/G, SEQ ID NO: 1) substrate cleavage site as the 27 kDaNIa proteinase.

In one embodiment, polypeptides of the present invention comprise aproteinase having a molecular weight of 27 kDa and reduced self-cleavageactivity as compared to the wild-type 27 kDa NIa proteinase. In anotherembodiment, the polypeptides of the present invention comprise themutant form of the 27 kDa NIa proteinase having an amino acid sequencein which the residue corresponding to Ser219 of the 27 kDa NIaproteinase is replaced with another residue, preferably Asn.

Mutant Forms and Allelic Forms of Proteinases.

The present invention also include mutant forms of proteinases havingthe same substrate cleavage activity as the wild-type 27 kDa NIaproteinase and having reduced self-cleavage activity. As discussedabove, to construct mutant forms of proteinases, the nucleic acidencoding the wild-type proteinase can be used as a starting point andmodified to form the desired mutants. For example, in the preferredembodiment, the nucleic acid sequence encoding the wild-type 27 kDa NIaproteinase is mutated such that Ser corresponding to position 219 in theencoded amino acid sequence is replaced with another amino acid,preferably Asn. Further, numerous methods are known to delete and mutatenucleic acid sequences that encode a polypeptide and to confirm thefunction of the polypeptides encoded by these deleted or mutatedsequences. Accordingly, the invention also provides mutated or deletedversion of a proteinase that has the same substrate cleavage activity asthe wild-type 27 kDa NIa proteinase.

Conservative variants of the wild-type 27 kDa NIa proteinases or itsnaturally occurring isoforms and homologs are encompassed. Suchconservative mutations have been discussed under the previous section.The present invention also contemplates conservative variants that donot affect the ability of the proteinase to recognize and cleave theheptapeptide sequence E-X-X-Y-X-Q↓S/G (SEQ ID NO: 1). The presentinvention includes 27 kDa NIa proteinase with altered overall charge,structure, hydrophobic/hydrophilic properties by amino acidsubstitutions, insertions, or deletions but still possess the ability torecognize and cleave the heptapeptide.

The present invention also contemplates naturally occurring allelicvariants of the proteinases having the same substrate cleavage activityas the 27 kDa NIa proteinase. In a preferred embodiment, allelicvariants even though possessing a slightly different amino acid sequencethan the naturally occurring wild-type 27 kDa proteinase will have therequisite ability to recognize and cleave the heptapeptide sequenceE-X-X-Y-X-Q↓S/G (SEQ ID NO: 1).

Preferably, proteinases having the same substrate cleavage activity asthe wild-type 27 kDa NIa proteinase and at least about 70% sequenceidentity, more preferably, at least about 80% sequence identity, evenmore preferably, at least about 85% sequence identity, yet morepreferably at least about 90% sequence identity, and most preferably atleast about 95% sequence identity to a wild-type 27 kDa NIa proteinaseor other naturally occurring isoforms having the same substrate cleavageactivity.

Numerous methods for determining percent homology are known in the art.One preferred method is to use version 6.0 of the GAP computer programfor making sequence comparisons. The program is available from theUniversity of Wisconsin Genetics Computer Group and utilizes thealignment method of Needleman and Wunsch, J. Mol. Biol. 48, 443, 1970,as revised by Smith and Waterman Adv. Appl. Math. 2, 482, 1981. Numerousmethods for determining percent identity are also known in the art, anda preferred method is to use the FASTA computer program, which is alsoavailable from the University of Wisconsin Genetics Computer Group.

Fusion Proteins.

The present invention also provides fusion proteins comprising aproteinase having the same substrate cleavage activity as the 27 kDa NIaproteinase fused to a heterologous protein or polypeptide. In oneembodiment, the fusion proteins of the present invention comprise aproteinase having a molecular weight of about 27 kDa and reducedself-clevage activity as compared to its wild-type proteinase fused to aheterologous protein. In another embodiment, the fusion proteins of thepresent invention comprise a mutant 27 kDa NIa proteinase having anamino acid sequence in which the residue corresponding to Ser219 of the27 kDa NIa proteinase is replaced with another residue, preferably Asn,fused to a heterologous protein.

As discussed earlier, various heterologous proteins may be fused to theproteinase of the present invention (see below also).

Compositions.

The present invention also provides compositions comprising an isolatedproteinase having the same substrate cleavage activity as the wild-type27 kDa NIa proteinase and a carrier. The composition may comprise a dryformulation or an aqueous solution. The carrier may be any compound thatdoes not affect the substrate cleavage activity of the proteinase.Carrier could be a diluent, an excipient, or even a stabilizer. Aspecific example of a carrier could be buffer or water, which does notaffect the stability of the proteinase.

Uses of Proteinases with Identical Substrate Cleavage Activity as the 27kDa NIa Proteinase.

The present invention also provides methods of using proteinases withthe same substrate cleavage activity as the 27 kDa NIa proteinase.Proteinases of the present invention can be used to cleave polypeptidescomprising the heptapeptide sequence E-X-X-Y-X-Q↓S/G (SEQ ID NO: 1). Themutant forms of the 27 kDa NIa proteinase with decreased self cleavageactivity are more stable than the wild-type proteinase and have a longershelf-life.

As discussed earlier, Parks et al. (1994) and Johnson et al., U.S. Pat.No. 5,532,142, disclose the use of the 27 kDa NIa proteinase as a toolfor purifying and obtaining large quantities of desired proteins. Asshown by Parks et al. and Johnson at al., to obtain large quantifies ofa desired protein, the protein is fused to a carrier protein and asubstrate cleavage site recognized by the 27 kDa NIa proteinase isinserted between the two proteins. The 27 kDa NIa proteinase is selectedfor separating the carrier protein from the desired protein because the27 kDa NIa proteinase exhibits unique characteristics. Unlike otherproteinases, the 27 kDa proteinase exhibits high specificity,insensitivity to many proteinase inhibitors used in proteinpurification, and efficient cleavage under broad range of temperatures(Polayes et al., 1994). Moreover, the protein of interest can be easilyseparated from the carrier peptide and the 27 kDa proteinase.

The present invention provides mutant forms of the 27 kDa NIa proteinasewith the same substrate activity as the wild-type proteinase and withdecreased self-cleavage activity. The mutant 27 kDa NIa proteinases ofthe present invention are also useful as tools for purifying andobtaining large quantities of desired proteins.

Proteins or Polypeptides that Form Inclusion Bodies.

Proteins, polypeptides, fusion proteins or polypeptides that forminclusion bodies in cells are also encompassed by the presentapplication. Such proteins, polypeptides, or fusion proteins, eitherproduced by recombinant means or present in their native source, arethen purified by the purification method provided by the presentinvention, discussed below.

In a preferred embodiment, the fusion proteins comprising a protein ofinterest is fused to a carrier protein or fusion partner thatfacilitates its isolation. Examples of carrier proteins are not limitedto any particular protein, but may be selected from a wide variety ofproteins such as beta galactosidase, ubiquitin, glutathioneS-transferase, alkaline phosphatase, maltose binding protein, Protein A,polyhistidines, monoclonal antibody epitopes and so forth. Carrierproteins typically will be selected on the basis of characteristicscontributing to easy isolation, most desirable being those that arereadily secreted by the microorganisms or which have some property orfeature which facilitates isolation and purification of the protein.Glutathione S-transferase, maltose binding protein and polyhistidinesequences, for example, are generally preferred because there arereadily available affinity columns to which they can be bound andeluted. Other suitable fusion partners include antigenic tags thatreadily bind to corresponding antibodies or proteins that have specialaffinity properties, for example, selective binding to particularmetals, as with polyhistidine peptide binding to nickel.

3. Recombinant Production of Proteinases

Vectors and Expression vectors.

The present invention provides vectors and expression vectors comprisinga nucleic acid encoding a proteinase having the same substrate cleavageactivity as the wild-type 27 kDa NIa proteinase. In a preferredembodiment, the vectors or expression vectors comprise a nucleic acidencoding a proteinase having a molecular weight of about 27 kDa andreduced self-cleavage activity as compared to its wild-type proteinase.In a more preferred embodiment, the vectors or expression vectorscomprise the nucleic acid encoding a mutant form of the 27 kDa NIaproteinase having the same substrate cleavage activity as its wild-type27 kDa NIa proteinase and reduced self-cleavage activity as compared toits wild-type proteinase.

The present invention also provides vectors and expression vectorscontaining the nucleic acids encoding fusion proteins and encoding anyprotein that forms inclusion bodies in cells. Preferably, the fusionproteins comprise a proteinase having the same substrate cleavageactivity as the 27 kDa NIa proteinase and a heterologous protein. Morepreferably, the proteinase of the fusion protein is a mutant form of 27kDa NIa proteinase having reduced self-cleavage activity and has anamino acid sequence in which the residue corresponding to position 240is replaced with another amino acid, preferably Asn.

Vectors or cassettes useful for the transformation and transfection ofsuitable host cells are well known in the art. Typically, the vectors orcassettes contain sequences directing transcription and/or translationof the relevant gene, a selectable marker, and sequences allowingautonomous replication or chromosomal integration. In an autonomouslyreplicating vector, i.e., a vector which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the host cell, is integrated into thegenome and replicated together with the chromosome(s) into which it hasbeen integrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused.

Suitable vectors for the present invention comprise a region 5′ of thegene which harbors transcriptional initiation regulation or control anda region 3′ of the DNA fragment which regulates transcriptionaltermination. It is most preferred when both regulatory regions arederived from nucleic acids homologous to the transformed host cell,although it is to be understood that such regulatory regions need not bederived from the nucleic acids native to the specific species chosen asa production host.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding theproteinase or protein formed in inclusion bodies, vectors based on SV40or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the proteinase or protein that formsinclusion bodies in a cell. For example, when large quantities of theprotein are needed for the induction of antibodies or for use as a toolin the purification of proteins, vectors which direct high levelexpression of fusion proteins that are readily purified may be used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as BLUESCRIPT (Stratagene), inwhich the sequence encoding the proteinase may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced. An example of such a vector include pIN vectors (Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509).

pGEX vectors (Promega, Madison, Wis.) are used to express foreignpolypeptides as fusion proteins with a heterologous protein such asglutathione S-transferase (GST). In general, fusion proteins are solubleand can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to include aprotease cleavage site, such as the substrate cleavage site of the 27kDa NIa proteinase, so that the purified polypeptide of interest can beeasily released from the GST moiety. The fusion proteins may alsocomprise a preferred proteinase or any protein that forms inclusionbodies in a cell and a carrier peptide or protein, such as the His tag,for affinity purification of the proteinase or protein. A furtherdiscussion of vectors which contain fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used (Grant et al. (1987) Methods Enzymol.153:516-544). In cases where plant expression vectors are used, theexpression of sequences encoding the proteinase or protein that formsinclusion bodies in a cell may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CAMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G, et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. at al. (1991) Results Probi. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express the proteinase or proteinthat forms inclusion bodies in a cell. For example, in one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes in Spodoptera frugiperda cells or inTrichoplusia larvae. The sequences encoding the protein may be clonedinto a non-essential region of the virus, such as the polyhedrin gene,and placed under control of the polyhedrin promoter. Successfulinsertion of the protein will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which PLBP may be expressed (Engelhard, E. K. el.al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding the proteinase or protein that form inclusionbodies in a cell may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing PLBP in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding the proteinase or protein formed ininclusion bodies. Such signals include the ATG initiation codon andadjacent sequences. In cases where sequences encoding a protein, itsinitiation codon, and upstream sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational regulatory signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

Host Cells.

The present invention also provides host cells, comprising a nucleicacid sequence encoding a proteinase as described above or a protein thatforms inclusion bodies in a cell, which are used in the recombinantproduction of the encoding the proteinase or protein. A vectorcomprising the nucleic acid sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The choice of a host cell will to a large extentdepend upon the gene encoding the polypeptide and its source. The hostcell may be a unicellular microorganism, e.g., a prokaryote, or anon-unicellular microorganism, e.g., a eukaryote. The host cell may be aeukaryote selected from the group consisting of mammalian cell, insectcell, plant cell or fungal cell.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus lichenifonnis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is E. Coli. Variousstrains of E. coli (e.g., HB101, DH5, DH10, and MC1061) are well-knownas host cells in the field of biotechnology.

Mammalian cells, such as Chinese hamster ovary cells (CHO) or 3T3 cellsmay be used in the present invention. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines, are the monkeyCOS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss,Balb-c or NIH mice, BHK or HaK hamster cell lines.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention (Miller etal., 1986 Genetic Engineering 8:277-298).

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

Host cells transformed with nucleotide sequences encoding proteinase andprotein that form inclusion bodies may be cultured under conditionssuitable for the expression and recovery of the proteinase from cellculture. The protein produced by a transformed cell may be secreted,contained intracellularly, or contained with the inclusion factordepending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containingpolynucleotides which encode the protein may be designed to containsignal sequences which direct secretion of the protein through aprokaryotic or eukaryotic cell membrane.

Other constructions may be used to join sequences encoding the proteinto nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitroger, San Diego, Calif.) or the 27 kDaNIa proteinase cleavage site between the purification domain and theprotein may be used to facilitate purification. One such expressionvector provides for expression of a fusion protein containing theprotein and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying from the fusion protein.

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” faun of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC; 10801 University Boulevard, Manassas, Va.,20110-2209) and may be chosen to ensure the correct modification andprocessing of the foreign protein.

For long-term, high-yield production of proteinases or proteins thatform inclusion bodies in a cell, stable expression is preferred. Forexample, cell lines which stably express proteinase or proteins thatform inclusion bodies may be transformed using expression vectors whichmay contain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol.150:1-14); and als or pat, which confers resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hailman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, -glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding a proteinaseor a protein that forms inclusion bodies in a cell, is inserted within amarker gene sequence, transformed cells containing sequences encodingthe protein can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with a sequenceencoding protein under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicatesexpression of the tandem gene as well. Alternatively, host cells whichcontain the nucleic acid sequence encoding the protein and express theprotein may be identified by a variety of procedures known to those ofskill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridizations and substrate cleavage assay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of nucleic acid orprotein.

Production of Protein from Host Cells.

The present invention also provides methods for producing a proteinaseor a protein that forms inclusion body in a cell comprising (a)cultivating the host cell under conditions that allow expression of theprotein; and (b) recovering the protein. The proteinase of the presentinvention has a substrate cleavage activity that is identical to that ofthe 27 kDa NIa proteinase. Preferably, the proteinase has a molecularweight of about 27 kDa and a reduced self-cleavage activity as comparedto its wild-type proteinase. More preferably, the proteinase is a mutantform of the 27 kDa NIa proteinase, and comprises an amino acid sequencein which the residue corresponding to position 219 of the wild-type 27kDa NIa is replaced with another amino acid, preferably Asn.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of the proteinsof the present invention using methods known in the art. For example,the cell may be cultivated by shake-flask cultivation, small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentersperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the protein is secreted into the nutrient medium, thepolypeptide can be recovered directly from the medium. If the protein isnot secreted, it can be recovered from cell lysates as described below

The proteins may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the protein.

The resulting protein may be recovered by methods described below.

4. Purification of Proteins

General Procedure for Purification of Proteins.

The proteins of the present invention produced from host cells may berecovered by methods known in the art. For example, the polypeptide maybe recovered from the nutrient medium by conventional proceduresincluding, but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The proteins of the present invention may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

In one embodiment of the invention, when the protein is secreted intothe culture medium, the step of collecting the conditioned culturemedium is followed by the step of purifying the protein. The protein inthe collected medium may be purified by any method known in the art,such as selective precipitation, affinity chromatography, dialysis,immunoprecipitation, ion-exchange chromatography, size-exclusionchromatography, hydrophobic interaction chromatography, orreversed-phase chromatography (Guide to Protein Purification, in Methodsin Enzymology 182 (Murray P. Deutscher ed., 1990), Remington: TheScience and Practice of Pharmacy 534-58 (Alfonso R. Gennaro ed., 19thed. 1995)). Chromatography can be carried out using conventional columnsor by HPLC (high performance liquid chromatography) or FPLC (fastprotein liquid chromatography).

In another embodiment of the invention, the protein is purified byanion-exchange chromatography. Methods of purifying proteins usinganion-exchange chromatography are well known to those skilled in the art(See, e.g., Edward R. Rossomando, Ion-Exchange Chromatography, in Guideto Protein Purification, Methods of Enzymology 182, 309-16 (Murray P.Deutscher ed., 1990)). In an alternate embodiment of the invention, theprotein is purified by affinity chromatography over a protamine-agarosecolumn, such as a protamine-Sepharose® (Pharmacia-LKB) column. Methodsof purifying the proteins of the present invention usingprotamine-agarose columns are known to those skilled in the art (Dempfleand Heene, Thromb. Res. 46, 19 (1987)). Preferably, the proteins of thepresent invention are purified by affinity chromatography using a columncontaining Ni-NTA Superflow resin (nickel-nitilotriacetic acid-agarose,Qiagen)

In an alternate embodiment of the invention, the protein is purified byimmunoaffinity chromatography using polyclonal or monoclonal antibodiesraised against the intact protein or peptides thereof of the presentinvention. Methods of producing and using polyclonal and monoclonalantibodies are well known in the art (Ed Harlow & David Lane,Antibodies: A Laboratory Manual (1988); Norman A. Staines, MonoclonalAntibodies, in Biochemical Research Techniques: A Practical Introduction(John M. Wrigglesworth Ed., 1983)). Likewise, methods of performingaffinity chromatography using polyclonal and monoclonal antibodies arealso well known in the art. (Ed Harlow & David Lane, Antibodies: ALaboratory Manual (1988); Steven Ostrove, Affinity Chromatography.General Methods, in Guide to Protein Purification, Methods of Enzymology182, 357-71 (Murray P. Deutscher ed., 1990)).

In another embodiment of the invention, when the protein is not secretedinto the culture medium, it is necessary to remove the protein from thecell by lysing the cells using methods well known in the art such assonication or freeze-thawing, followed by isolating the protein from thecell extract. The cell extract may be prepared or incubated in thepresence of cell protease inhibitors. The plant virus cleavage site isnot susceptible to proteolysis by ordinary cell proteases. The plantvirus proteinase itself is unaffected by ordinary protease inhibitors sothat such inhibitors may be added in amounts sufficient to inactivatecell proteases. Typical and commonly used cell protease inhibitorsinclude leupeptin, pepstatin A, PMSF, E-64, TLCK, bestatin andaprotinin. However, any of a number of proteinase inhibitors may beemployed so long as they are not inhibitors of the proteinase used torelease a foreign protein from the carrier protein. The practitionerwill typically culture in a media or grow in an environment suitable forthe host selected, prepare cell extract, then add appropriate cellprotease inhibitors. The desired protein may be purified using standardprocedures such as chromatography, electrophoresis or density gradientcentrifugation.

The above methods are also applicable to purifying proteins from theirnative source.

Procedure for Purification of Proteins that Form Inclusion Bodies.

U.S. Pat. No. 5,989,554 provides a general method for isolating andpurifying proteins that form inclusion bodies in cells. First, the cellsare lysed by enzymatic or mechanical means in a buffer. The preferredmethod is sonication, although any other lysis method will work, as longas lysis is complete and DNA and RNA are sufficiently fragmented so asnot to pellet upon centrifugation. Preferred buffers contain Tris bufferat pH 7-8, isotonic saline, and dithiothreitol (DTT) to maintain allcell proteins in a reduced state. After sonication, detergent is addedto the mixture to solubilize most lipids and proteins, and the mixtureis centrifuged; it is preferred to use a centrifuge speed of greaterthan 10,000 g for 10 minutes. The desired protein is then found in thepellet fraction at a high degree of purity.

Higher purity is usually obtained by washing the pellet in a second washsolution, often containing a different agent or detergent. Washing isaccomplished by resuspending the pellet in the fresh buffer followed bycentrifugation as above. The preferred first detergent is sodiumdeoxycholate (NaDOC), and the second preferred detergent is TritonX-100.

After the detergent washes, the pellet can be washed either with theabove buffer or with phosphate-buffered saline (PBS) to remove tracedetergent, then resuspended in a volume of a desired buffer for storageor use in any of the ways described elsewhere, including immunization.

The proteins purified by the above method are ready for use in any orall of the applications contemplated in the invention, including but notlimited to the following; immunization of animals, use as an adjuvant,coupling to other ligands, use as a protease inhibitor, immobilizationon hydrophobic surfaces, use as an enzyme substrate, and use in peptideproduction after cleavage.

As discussed earlier, this method is labor-intensive and does notprovide large quantities of active protein.

Novel Method for Purifying Proteins that Form Inclusion Bodies.

The present invention is based on the development of a single-columnpurification method for preparing milligram quantities of >95% pure,active 27 kDa NIa proteinase. The method comprises the following steps:

-   -   a) obtaining cells expressing the polypeptide;    -   b) lysing the cells;    -   c) pelleting inclusion bodies by centrifugation of the lysed        cells;    -   d) centrifuging the inclusion bodies;    -   e) solubilizing the inclusion bodies in buffer containing        solubilization agent;    -   f) centrifuging the solubilized inclusion bodies to obtain a        supernatant containing the polypeptide;    -   g) loading the supernatant over a single denaturing column;    -   h) collecting the proteinase; and    -   i) renaturing the collected polypeptide.        Preferably, the cells are lysed by incubating for 30 min at        4° C. in 50 mM Tris-Ci, pH 8.0, 300 mM NaCl, 500 μg/ml lysozyme,        200 μg/ml DNase I, and protease inhibitors (such as PMSF and        leupeptin); followed by three freeze-thaw cycles; addition of        Triton X-100 to a final concentration of 1%; and vortexing.        Preferably, following lysis the inclusion bodies are pelleted by        centrifugation at 10,000×g. Preferably, the inclusion bodies are        then solubilized with 6 M GuHCl, 100 mM NaH₂PO₄ and 10 mM        Tris-Cl, pH 8.0. Preferably, before loading on the denaturing        column the solubilized inclusion bodies are centrifuged for 20        minutes at 15,000×g. Preferably, the denaturing column contains        Ni-NTA (nicke-nitilotiacetic acid-agarose) Superflow resin        (Qiagen). Preferably, the proteinase is eluted with 6 M urea,        100 mM NaH₂PO₄, and 10 mM Tris-Cl, pH 4.5, and the collected        proteinase fraction is adjusted with 10 N NaOH to pH 8.5, and        renatured during dialysis for 4-8 hours in storage buffer (100        mM Tris-Cl, pH 8.5, 500 mM NaCl, 10% Glycerol, 5 mM DTT, 0.5 mM        EDTA). Preferably, as a final step, the dialyzed proteinase        fraction is centrifuged for 30 minutes at 15,000×g at 4° C. to        collect the supernatant containing the active renatured        proteinase.

Other proteinase inhibitors, methods for lysing the cells, buffers,solubilizing agents, and methods of renaturing the purified protein arewell known to the skilled artisan, and some are described in theprevious sections of the specification. It is also pointed out that anyhost cell that expresses large quantities of the protein of interest inthe form of inclusion bodies or any cell that endogenously expresses theprotein in the form of inclusion bodies are useful for purifying largequantities of the protein of interest.

In a preferred embodiment, the protein to be isolated in largequantities is expressed in E. coli as a fusion protein comprising inaddition to the protein, a proteinase cleavage site, and a fusionpartner. Proteinase cleavage sites, as well as fusion partner or carrierprotein, are well known to the skilled artisan and have been describedin detail in other sections of the present specification. A preferredproteinase cleavage site is the site recognized by the 27 kDa NIaproteinase and the preferred carrier protein is six His tags. Thehistidines enables binding to the Ni-NTA column. The 27 kDa NIaproteinase, unlike other proteinases, exhibits high specificity,insensitivity to many proteinase inhibitors used in proteinpurification, and efficient cleavage under broad range of temperatures(Polayes et al., 1994). Moreover, the protein to be purified is easilyseparated from the carrier peptide and the 27 kDa NIa proteinase.

In light of the foregoing general discussion, the specific examplespresented below are illustrative only and are not intended to limit thescope of the invention. Other generic and specific configurations willbe apparent to those persons skilled in the art.

EXAMPLES Example 1 Cloning

A wild type 27 kDa NIa nucleic acid was generated by PCR using pTL-5495(ATCC 45036) as the template, 5TEVP1: 5′-CAT CAG CGG GCC ATG GCT GAA AGCTTG TTT AAG-3′ (SEQ ID NO: 2) as the 5′ primer, and 3TEVH1: 5′-CTG ATGCAC GGA TCC TCA TTA ATG GTG ATG GTG ATG GTG CAA TTG CGA GTA GAC TAA TTCACT CAT G-3′ (SEQ ID NO: 3) as the 3′ primer. This nucleic acidtranslates to a proteinase with a C-terminal His₆tag. The mutant 27 kDaNIa nucleic acid was generated by sequential PCR reactions usingpTL-5495 as the template for the first reaction with 5TEVP1 as the 5′primer and TEVPSN: 5′-GAG TTG AGT TGC TTC TTT GAC TGG CTG AAA GGG TTCTTC AGG TTT GTT CAT GAA AAC TTT GTG GCC-3′ (SEQ ID NO: 4) as the 3′primer to introduce the S219N mutation at the internal self-cleavagesite. The resultant PCR product was used as the template in a second PCRreaction using the 5′ primer 5TEVP1 and the 3′ primer 3TEVH1. Thenucleic acids were ligated into the pET15b expression vector (Novagen)using the NcoI and BamHI sites and transformed into E. coli DH5a(GibcoBRL) competent cells. Plasmids, pTPWT (wild type 27 kDa NIaproteinase) and pTPSN (mutant foim with Ser to Asn mutation), weresequenced for accuracy and transformed into BL21 (DE3) (Novagen)competent cells for expression.

Example 2 Proteinase Expression and Purification

The wild-type and mutant form 27 kDa NIa proteinases were expressed inBL21 (DE3) cells grown at 37° C. in Luria Broth with 100 μM Ampicillinto optical density 600>0.7. Cultures were induced with 400 μM IPTGfor >4 hours. Cell pellets were harvested by centrifugation, resuspendedin 50 ml buffer containing 50 mM Tris-Cl, pH 8.0 and 300 mM NaCl perliter of cell culture, and stored at −80° C. Lysis and purification ofthe soluble fraction containing the 27 kDa NIa proteinase were performedas described by Parks et al. (1995) through the Ni-NTA agarosepurification step, except 10% glycerol, 300 mM NaCl, and 5 mM βME wereincluded in all buffers, and the Ni-NTA agarose column was washed withbuffer containing 10 mM imidazole and eluted with buffer containing 400mM imidazole.

For inclusion body purification, cell suspensions were thawed in coolwater and the buffer was adjusted to give a final concentration of 500μg/ml lysozyme (Sigma), 200 μg/ml DNASE I (Boehringer Mannheim), 50μg/ml PMSF, 10 μg/ml Leupeptin (Boehringer Mannheim), 20 mM MgSO₄, and 2mM CaCl₂. The cells were lysed as follows: rocking for 30 minutes at 4°C., followed by 3 freeze-thaw cycles, lysed the cells; adding Tritonx-100 to a final concentration of 1%; and vortexing. The inclusionbodies were pelleted by centrifugation at 10,000×g. Purified inclusionbodies were solubilized in a buffer containing 6 M GuHCl, 100 mM NaH₂PO₄and 10 mM Tris-Cl, pH 8.0, and stored at −80° C.

Denaturing column chromatography was done at 4° C. using 10 ml Ni-NTASuperflow resin (Qiagen) per liter cell culture. The column wasequilibrated with 10 column volumes (cv) of equilibration buffer (6MUrea, 100 mM NaH₂PO₄, and 10 mM Tris-Cl, pH 8.0). The solubilizedinclusion bodies were thawed in a 65° C. bath and centrifuged 20 minutesat 15,000×g at 4° C. The supernatant was loaded onto the column bygravity flow. The column was washed with 4 cv equilibration buffer, then6 cv wash buffer (6 M Urea, 100 mM NaH₂PO₄, and 10 mM Tris-Cl, pH 6.3).The proteinase was eluted in elution buffer (6 M Urea, 100 mM NaH₂PO₄,and 10 mM Tris-Cl, pH 4.5) with a 5 minute column incubation betweeneach fraction until a total of 6 fractions were collected. Fractionscontaining the 27 kDa NIa proteinase were pooled, adjusted to pH 8.5with 10 N NaOH, dialyzed 4-8 hours in storage buffer (100 mM Tris-Cl, pH8.5, 500 mM NaCl, 10% Glycerol, 5 mM DTT, and 0.5 mM EDTA), andcentrifuged 30 minutes at 15,000×g at 4° C. The supernatant, containingactive, renatured 27 kDa NIa proteinase was separated from the pellet,containing precipitated 27 kDa NIa proteinase, and both were stored at−80° C. The pellet is successively resuspended in Equilibration Bufferand redialyzed to obtain more renatured, active proteinase as needed.

The 27 kDa Ma proteinase preparations were quantitated using theBradford Assay (Biorad) using BSA as a standard. The yield of active 27kDa NIa proteinase from the soluble preparation was estimated to be lessthan 10% of the total protein as this sample was not assayed foractivity. For the insoluble preparation, the active 27 kDa NIa yield isreported as milligrams of proteinase obtained from the firstrenaturation of the eluate from the Ni-NTA agarose column.

Example 3 Activity Assays

A 17 kDa substrate containing the target cleavage siteGlu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 5) produces a ˜15 kDa peptidewhen cleaved with commercially available 27 kDa NIa proteinase(GibcoBRL). Substrate cleavage activity was assayed in 30 μl reactionswith 100, 250, or 500 μM substrate and 0.75 μg proteinase in assaybuffer (50 mM Tris-Cl, pH 8.0, 1 mM DTT, and 0.5 mM EDTA) incubated at30° C. for 1 hour. Samples were electrophoresed in 15% SDS-PAGE gels,stained with Coomassie Blue, and protein bands were quantitated using agel documentation system (Kodak). The net intensities of full-length andcleaved substrate bands were used to calculate the specific activity forthe substrate at each substrate concentration.

Self-cleavage activity was assayed in 40 μl reactions containing 7.5 μg27 kDa Ma proteinase in 50 mM Tris-Cl, pH 8.0, 1 mM DTT, and 0.5 mMEDTA, incubated at 4° C., −20° C., and −80° C. At 0.5, 1, 2, and 5.5weeks, 10 μl sample was removed and boiled with denaturing SDS-PAGEloading buffer, loaded onto 15% SDS-PAGE gels, and quantitated as perabove. The net intensities of full-length and cleaved proteinase bandswere used to calculate the percent proteinase cleaved at each timepoint.

Results

pTPWT and pTPSN encode peptides with respective molecular weights of28,563 Daltons (about 28.5 kDa) and 28,590 Daltons (about 28.5 kDa).During cloning, the proteinase N-terminus was altered from wild typeGly-Glu-Ser- to Met-Ala-Glu-Ser-(SEQ ID NO: 6) and a-Leu-His-His-His-His-His-His (SEQ ID NO: 7) tag was added to theC-terminus.

Soluble preparations of 27 kDa NIa proteinase yielded minimal quantitiesof proteinase (Table 1). 27 kDa NIa proteinase detected as a band incell lysate is missing in clarified lysate but present in the inclusionbody preparation (FIG. 1, lanes 1-3 and 7-9). Ni-NTA agarosepurification under denaturing conditions yields purer proteinase thanpurification under soluble conditions (FIG. 1, lanes 4, 5, 10, and 11).In samples containing 4.5 μg total protein, a single contaminant isdetected in renatured 27 kDa NIa proteinase after the denaturingpurification indicating >95% purity (FIG. 1, lanes 13-15). Final yieldsof >10 mg renatured, active 27 kDa NIa proteinase were regularlyobtained after a single renaturation step for both TPWT and TPSN (Table1). Final concentrations of several renatured preps indicate a maximumsolubility of ˜1.5 mg/ml (data not shown).

TABLE 1 Purification Yields for Soluble vs Insoluble Procedures (perliter cell culture). Total protein Total active Ni-NTA Ni-NTA-pure rTEVP% load (mg) protein (mg) (mg)* yield** TPWT Soluble prep 977 4.08 <0.410.04 Insoluble prep 80 30.29 >10.96 1.04 TPSN Soluble prep 946 3.67<0.37 0.04 Insoluble prep 81 32.35 >12.84 1.25 *Active rTEVP for thesoluble preps are estimated to be <10% of total purified protein. Datareported for insoluble preps is reported as greater than the quantity ofprotein obtained from a single round of renaturation. **% yield activerTEVP from total cellular protein Note: Yields of total protein, active27 kDa NIa proteinase, and overall percent yield are shown for both thesoluble and insoluble purification protocols. Protein quantitation ateach purification step was estimated using the Bradford assay with a BSAstandard curve.

The specific activity for substrate cleavage is defined as the picomolesubstrate cleaved per picomole 27 kDa NIa proteinase in 1 hour at 30° C.The data indicate that TPWT and TPSN have similar activity to each otherand to the commercially available proteinase at all substrateconcentrations tested (FIG. 2).

Both the commercially available 27 kDa NIa proteinase and purified TPWTcontain the truncated self-cleavage product in the final preparation,while the TPSN preparation contains only full-length proteinase (FIG. 1,lanes 13-15). The self-cleavage activity of TPWT is similar to thecommercially available proteinase at 4° C. and −20° C. TPSN has reducedself-cleavage activity at 4° C., and no self-cleavage activity at −20°C. (FIGS. 3, 4 a, and 4 b). None of the proteinase samples testedexhibited significant self-cleavage activity at −80° C. during our 5.5week assay (data not shown).

Conclusions

The present invention shows that wild type, as well as mutant,histidine-tagged 27 kDa NIa constructs can be induced to express >95% ofthe proteinase in the insoluble fraction (FIG. 1). The present inventionprovides a novel method of denaturing purification of the insolublefraction followed by renaturation of the peptide that yields up to 10times as much active 27 kDa NIa proteinase as the soluble preparationreported by Parks et al (1995). This allows for stock preparationsof >95% pure, active 27 kDa NIa proteinase to be made for general use.

Parks et al. (1995) has shown that the truncated form of 27 kDa NIaproteinase has significantly less substrate cleavage activity than thefull-length form. The proteinase continues to cleave itself over timewhen stored at 4° C. and −20° C., potentially reducing the quantity offully active enzyme in an 27 kDa NIa proteinase stock over time.Self-cleavage appears to be arrested at −80° C.

The Ser219→Asn mutation in the mutant form of the 27 kDa NIa proteinase,provided by the present invention, significantly inhibits self-cleavageactivity, allowing for increased yields of full-length, fully active 27kDa NIa proteinase, from either soluble or insoluble preps. The presentinvention also permits long-term storage of 27 kDa NIa stocks at −20°C., and short-term storage at 4° C.

The 27 kDa proteinase and its mutant form are valuable tools for proteinpurification protocols because of its target site specificity and itsactivity under a wide variety of conditions. The present invention byutilizing a denaturing preparation of the TPSN mutant, enablesproduction of large stocks of rTEVP with consistent activitycharacteristics.

It should be understood that the foregoing discussion and examplesmerely present a detailed description of certain preferred embodiments.It therefore should be apparent to those of ordinary skill in the artthat various modifications and equivalents can be made without departingfrom the spirit and scope of the invention. All journal articles, otherreferences, patents, and patent applications that are identified in thispatent application are incorporated by reference in their entirety.

REFERENCES

Allison, R., Johnston, R. E., and Dougherty, W. G. (1986) The NucleotideSequence of the Coding Region of the Tobacco Etch Virus Genomic RNA:Evidence for the Synthesis of a Single Polyprotein. Virology 154, 9-20

Argos, P., Kamer, P., Nicklin, M. J. H., and Wimmer, E. (1984).Similarity in gene organization and homology between proteins of animalpicornaviruses and plant comoviruses suggest common ancestry of thesevirus families. Nucleic Acids Res. 12, 7251-7267.

Bazan, J. F., and Fletterick, R. J. (1990). Structural and catalyticmodels of trypsin-like viral proteases. Semin. Virol. 1, 311-322.

Carrington, J. C. and Dougherty, W. G. (1988) A Viral Cleavage SiteCassette: Identification of Amino Acid Sequences Required for TobaccoEtch Virus Polyprotein Processing. Biochem 85, 3391-3395

Carrington, J. C., Cary, S. M., Parks, T. D., and Dougherty, W. B.(1989) A Second Proteinase Encoded by a Plant Potyvirus Genome. EMBO J.8, 365-370

Dougherty, W. G., Parks T. D., Cary, S. M, Bazan, J. F. and Fletterick,R. J. (1989b). Characterization of the catalytic residues of the tobaccoetch virus 49-kDa proteinase. Virology 172, 302-310.

Dougherty, W. G., Cary, S. M. and Parks T. D. (1989a). Molecular geneticanalysis of a plant virus polyprotein cleavage site: A model. Virology171, 356-364.

Dougherty, W. G., and Parks T. D. (1989). Molecular genetic andbio-chemical evidence for the involvement of the heptapeptide cleavagesequence in determining the reaction profile at two tobacco etch viruscleavage sites in cell-free assays. Virology 172, 145-155.

Dougherty, W. G., and Parks T. D. (1991). Post-translational processingof the tobacco etch virus 49-kDa small nuclear inclusion polyprotein:Identification of an internal cleavage site and delimitation of Vpg andproteinase domains. Virology 183, 449-456.

Dougherty, W. G. and Hiebert, E. (1980) Translation of Potyvirus RNA ina Rabbit Reticulocyte Lysate: Identification of Nuclear InclusionProteins as Products of Tobacco Etch Virus RNA Translation andCylindrical Inclusion Protein as a Product of the Potyvirus Genome. Vir104, 174-182

Dougherty, W. G. and Parks, T. D. (1989a) Molecular Genetic andBiochemical Evidence for the Involvement of the Heptapeptide CleavageSequence in Determining the Reaction Profile at Two Tobacco Etch VirusCleavage Sites in Cell-Free Assays. Vir 172, 145-155

Dougherty, W. G., Carrington, J. C., Cary, S. M., and Parks, T. D.(1988) Biochemical and Mutational Analysis of a Plant Virus PolyproteinCleavage Site. EMBO 7, 1281-1287

Dougherty, W. G., Cary, S. M., and Parks, T. D. (1989b) MolecularGenetic Analysis of a Plant Virus Polyprotein Cleavage Site: A Model.Vir 171, 356-364

Krausslich, H. G., and Wimmer, E. (1988). Viral Proteinases. Annu. Rev.Biochem. 57, 701-754.

Lawson, M. A. and Semler, B. L. (1990) Picornavirus Protein Processing:Enzymes, Substrates, and Genetic Regulation. Curr. Topics Micro. Immun.161: 49-87

Lawson, M. A. and Semler, B. L. (1991) Alternate PoliovirusNon-Structural Protein Processing Cascades Generated by Primary Sites of3C Proteinase Cleavage. Virology 191: 309-320

Parks, T. D., Howard, E. D., Wolpert, T. J., Arp, D. J., and Dougherty,W. G. (1995) Expression and Purification of a Recombinant Tobacco EtchVirus Nia Proteinase: Biochemical Analyses of the Full-Length and aNaturally Occurring Truncated Proteinase Form. Vir 210, 194-201

Parks, T. D., Leuther, K. K., Howard, E. D., Johnston, S. A., andDougherty, W. G. (1994) Release of Proteins and Peptides from FusionProteins Using a Recombinant Plant Virus Proteinase. Anal. Biochem. 216,413-417

Polayes, D. A., Goldstein, A., Ward G., and Hughes, A. J. Jr. (1994) TEVProtease, Recombinant: A Site-Specific Protease for Efficient Cleavageof Affinity Tags from Expressed Proteins in “Focus” 16, #1, LifeTechnologies, Inc.

Taylor and Drickamer (1991) Carbohydrate-Recognition Domains as Toolsfor Rapid Purification of Recombinant Eukaryotic Proteins. BiochemistryJournal 99, 243-248

Verchot, J., Koonin, E N., and Carrington, J. C. (1991) The 35 kDaProtein from the N-terminus of the Potyviral Polyprotein Functions as aThird Virus-Encoded Proteinase. Virology 185, 60-69

The invention claimed:
 1. An isolated active mutant Potyvirus NIaproteinase that recognizes and cleaves a substrate cleavage site recitedin SEQ ID NO: 1, wherein the isolated active mutant proteinase hasreduced self-cleavage activity relative to self-cleavage activity of acorresponding wild-type proteinase, and wherein the isolated activemutant proteinase has a single amino acid change in its internalself-cleavage site at a residue corresponding to Ser 219 of a wild-typeTEV proteinase.
 2. The isolated active mutant proteinase of claim 1,wherein the mutant proteinase has a same or substantially same substratecleavage activity as the corresponding wild-type proteinase.
 3. Theisolated active mutant proteinase of claim 1, wherein the mutantproteinase has at least about 90% sequence identity to a wild-type 27kDa NIa proteinase.
 4. The isolated active mutant proteinase of claim 1,wherein the mutant proteinase has at least about 95% sequence identityto a wild-type 27 kDa NIa proteinase.
 5. A fusion protein comprising themutant proteinase of claim 1 fused to a heterologous polypeptide.
 6. Thefusion protein of claim 5, wherein the heterologous polypeptide consistsof six histidines.
 7. An isolated active mutant Potyvirus NIa proteinasecomprising an amino acid sequence in which a residue corresponding toSer 219 of a wild-type TEV proteinase is replaced with another residue,wherein the isolated active mutant proteinase has reduced self-cleavageactivity relative to self-cleavage activity of a corresponding wild-typeproteinase, and wherein the isolated active mutant proteinase recognizesand cleaves a substrate cleavage site recited in SEQ ID NO: 1.